RNA-seq of Drosophila melanogaster
Total RNA was quantified using a NanoDrop Spectrophotometer ND-1000 (NanoDrop Technologies, Inc.) and its integrity was assessed using a 2100 Bioanalyzer (Agilent Technologies). Libraries were generated from 250 ng of total RNA using the TruSeq stranded mRNA Sample Preparation Kit (Illumina), as per the manufacturers recommendations. Libraries were quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies) and the Kapa Illumina GA with Revised Primers-SYBR Fast Universal kit (Kapa Biosystems). The average size fragment was determined using a LabChip GX (PerkinElmer) instrument. For sequencing, the libraries were normalized and pooled at 3nM and then denatured in 0.05N NaOH and neutralized using the HT1 buffer. ExAMP was added to the mix following the manufacturers instructions. The pool now at 200pM was loaded on an Illumina cBot and the flow cell was run on a HiSeq 4000 for 2x100 cycles (paired-end mode).